Bioinformatics Characterization of Amine Oxidases
Lopes de Carvalho, Leonor (2018-12-14)
Lopes de Carvalho, Leonor
Åbo Akademi - Åbo Akademi University
14.12.2018
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Julkaisun pysyvä osoite on
https://urn.fi/URN:ISBN:978-952-12-3763-8
https://urn.fi/URN:ISBN:978-952-12-3763-8
Tiivistelmä
Amine oxidases catalyse the oxidative degradation of amines into the corresponding aldehyde, ammonia and hydrogen peroxide. They can be classified into different subfamilies based on the prosthetic group: (1) proteins with flavin adenine dinucleotide (FAD) make the FAD-dependent amine oxidase subfamily, which include monoamine oxidases (MAO) and polyamine oxidases (PAO) and (2) proteins with copper and quinocofactor make the other subfamily, which includes the copper-dependent amine oxidase (CAO) that have topaquinone (TPQ) as cofactor and the lysyl oxidases (LOX) that have the lysyl tyrosylquinone (LTQ) cofactor.
Most mammals have four CAO genes, each encoding a protein with a different function and substrate preference. The phylogenetic study with CAOs showed that these proteins can be classified into different subfamilies based on the conserved active site motif, X1-X2-Asn-Tyr-Asp. This classification is consistent with the different substrate preferences known for these proteins. Residue X2 can be used to distinguish AOC1 (X2=Tyr, diamine preference), AOC2 (X2=Gly, aromatic monoamine preference) and AOC3/4 (X2= Leu, aliphatic monoamine preference). The phylogenetic analysis led us to develop a novel classification system for the AOC3 and AOC4 proteins. These proteins can be classified based on the X1 residue from the motif, which is Leu in AOC3 and Met in AOC4.
When the N-glycosylations in hAOC1 were studied, it was found that the Asn110 site mainly contained oligomannosidic glycans. This site seems to be conserved in vertebrate CAOs and in hAOC1 it is located in a hydrophobic cleft on the surface. The attached glycans play a role in the correct folding, stabilization of the D2 domain, and protein secretion. Due to the high degree of conservation in the glycan composition, this N-glycosylation site likely plays a similar role in hAOC3 and porcine AOC1. The Nglycosylation sites at Asn168, Asn538 and Asn745 play a role in protein segregation and folding, and the Asn538 and Asn745 located near the inter-monomeric arms seem to be important for the dimer stability.
The interaction between hAOC3 and its leucocyte counter receptor, Siglec-9, is powerful tool for in vivo visualization of inflammation. In this thesis, the interactions between hAOC3 and a Siglec-9 derived peptide were analysed taking into account for the first time the glycosylations in hAOC3. The peptide binds in the active site of hAOC3 competing for the same binding site as semicarbazide and imidazole. Moreover, the predicted binding mode of the peptide is in accordance with PET studies using rodent, rabbit and pig AOC3 proteins.
The polyamine oxidases (PAO) belong to the FAD-dependent amine oxidases. PAOs are involved in the catabolism of spermidine and their acetyl. The PAO from Synechocystis sp. PCC 6803 (SynPAO) was characterized in this thesis. SynPAO is involved in the polyamine back-conversion pathway, with spermidine being the preferred substrate. The structural analysis together with the phylogenetic tree revealed that Gln94, Tyr403 and Thr440 are predicted to be key residues in the active site to SynPAO.
Most mammals have four CAO genes, each encoding a protein with a different function and substrate preference. The phylogenetic study with CAOs showed that these proteins can be classified into different subfamilies based on the conserved active site motif, X1-X2-Asn-Tyr-Asp. This classification is consistent with the different substrate preferences known for these proteins. Residue X2 can be used to distinguish AOC1 (X2=Tyr, diamine preference), AOC2 (X2=Gly, aromatic monoamine preference) and AOC3/4 (X2= Leu, aliphatic monoamine preference). The phylogenetic analysis led us to develop a novel classification system for the AOC3 and AOC4 proteins. These proteins can be classified based on the X1 residue from the motif, which is Leu in AOC3 and Met in AOC4.
When the N-glycosylations in hAOC1 were studied, it was found that the Asn110 site mainly contained oligomannosidic glycans. This site seems to be conserved in vertebrate CAOs and in hAOC1 it is located in a hydrophobic cleft on the surface. The attached glycans play a role in the correct folding, stabilization of the D2 domain, and protein secretion. Due to the high degree of conservation in the glycan composition, this N-glycosylation site likely plays a similar role in hAOC3 and porcine AOC1. The Nglycosylation sites at Asn168, Asn538 and Asn745 play a role in protein segregation and folding, and the Asn538 and Asn745 located near the inter-monomeric arms seem to be important for the dimer stability.
The interaction between hAOC3 and its leucocyte counter receptor, Siglec-9, is powerful tool for in vivo visualization of inflammation. In this thesis, the interactions between hAOC3 and a Siglec-9 derived peptide were analysed taking into account for the first time the glycosylations in hAOC3. The peptide binds in the active site of hAOC3 competing for the same binding site as semicarbazide and imidazole. Moreover, the predicted binding mode of the peptide is in accordance with PET studies using rodent, rabbit and pig AOC3 proteins.
The polyamine oxidases (PAO) belong to the FAD-dependent amine oxidases. PAOs are involved in the catabolism of spermidine and their acetyl. The PAO from Synechocystis sp. PCC 6803 (SynPAO) was characterized in this thesis. SynPAO is involved in the polyamine back-conversion pathway, with spermidine being the preferred substrate. The structural analysis together with the phylogenetic tree revealed that Gln94, Tyr403 and Thr440 are predicted to be key residues in the active site to SynPAO.